Stem cells are necessary to maintain homeostasis within adult tissues. These cells receive signals from their surrounding microenvironment or niche in order to function properly. Precise regulation of stem cells and their niches is vital to prevent depletion or overgrowth of the stem cell pool. Although many mammalian niches have been characterized, the Drosophila testis provides a unique and more accessible system to study the regulation of a stem cell niche in vivo. This well-defined niche consists of a cluster of non-dividing (or quiescent) somatic hub cells that signal to the attached germline and somatic stem cells. Damaging the niche or overexpressing genes that promote cell division in hub cells induces hub cell divisions, but also leads to the conversion of hub cells to somatic stem cells. This change in cell fate is accompanied by the formation of new niches, characterized by the presence of ectopic hubs each supporting active stem cells. The generation of excess niches in an adult tissue causes widespread tissue disruption over time, and is likely to underlie tumorigenesis quite generally. However, little is known about how this phenomenon is regulated, and understanding the underlying mechanisms is a long-standing goal of regenerative medicine. The goal of this proposal is to uncover the molecular mechanisms and cellular behaviors that are activated or deactivated upon tissue damage and regulate the ability of niche cells to lose quiescence and adopt a stem cell fate. Recently the highly conserved cell cycle inhibitor and tumor suppressor retinoblastoma homolog RBF was identified as a critical regulator of hub cell quiescence. Loss of RBF in the hub is sufficient to cause hub cell proliferation, conversion of hub cells into somatic stem cells, and ectopic niche formation. To understand what signaling pathways regulate RBF and its cell cycle interacting partners, a loss and gain-of-function screen of signaling components in the hub will be performed to further elucidate the mechanism. Live imaging of testes under loss of quiescence conditions has already revealed that converting hub cells migrate farther distances than their wild-type counterparts, and will allow cell fate changes to be tracked in real time. This technique will be further utilized to understand how ectopic niches form and how they acquire new stem cells. The proposed experiments will provide a model for understanding the regulation of niche cell quiescence and identity, which could be pertinent in other stem cell niches to prevent over-proliferation and metastases.